Method and device for detecting glucose concentration

ABSTRACT

A method and test device for determining the glucose concentration in a test sample containing from 1/2 to 10 percent glucose are disclosed. The method and test device involve impregnating the carrier with an enzymatic testing composition which includes glucose oxidase, peroxidase and a chromogen. The carrier is subsequently impregnated with from 0.5 to 1.5 percent by weight polystyrene. Contact of the carrier with a glucose-containing test sample produces a detectable response whereby the glucose concentration can be determined.

This application is a division of application Ser. No. 216,529, filedDec. 15, 1980, now U.S. Pat. No. 4,336,330.

BACKGROUND OF THE INVENTION

The detection of glucose in body fluids, as well as the determination ofits concentration therein, is of great importance for diabetic patientswho must control their diets so as to regulate their sugar intake andwho must frequently be guided in this regard by a regular check on urineglucose. The determination of glucose in urine is also important wherelarge numbers of people are screened to determine the incidence ofdiabetes among them.

Because early diagnosis and continued control are so important indiabetes, a glucose test, to be of greater value, must be convenientlyrapid, simple enough for the technician or patient to learn with ease,accurate enough to serve the clinician or patient, sensitive enough toreflect variations in the patient's condition, and specific for glucose.

Currently there are available sophisticated biochemical systems whichcan be incorporated into dry, dip-and-read reagent strip devices, usedin solution or suspension techniques, or in conjunction withspectrophotometics and other read-out systems.

These strips comprise a plastic strip, having at one end a carrierportion impregnated with an enzymatic testing composition which includesthe enzymes glucose oxidase and peroxidase and one or more indicatorcompounds as the principal active ingredients. Buffering agents may bepresent to keep the pH of the reactants at the site of reaction at apredetermined pH range. The strip utilizes an enzyme system wherein theglucose is a substrate for glucose oxidase. Glucose is oxidized togluconic acid with the concomitant formation of hydrogen peroxide.Indicator compounds present undergo color changes in the presence ofhydrogen peroxide and peroxidase. Various indicators can be usedincluding "benzidine-type" chromogens, e.g., benzidine, o-tolidine andtetramethylbenzidine and substituted aniline chromogens. A combinationof indicators can be utilized.

The glucose enzymatic test strips referred to above enable the assay ofglucose levels by measuring the rate of color change which the indicatorundergoes, i.e., by a rate reaction. The sample to be analyzed forglucose is contacted with the reagent-incorporated carrier portion bymomentarily immersing the carrier portion into the sample or by applyingan aliquot of the sample to the carrier portion and measuring theresponse after a set period of reaction time, by comparing any colorformed in the carrier portion with a standard color chart calibrated tovarious glucose concentrations.

The general principles of chemical reaction kinetics apply toenzyme-catalyzed reactions, but enzyme-catalyzed reactions also show adistinctive feature not usually observed in nonenzymatic reactions,saturation with substrate. The rate equation for reactions catalyzed byenzymes having a single substrate, e.g., glucose, is expressed by anequation known as the Michaelis-Menten equation. Under certain reactionconditions, the Michaelis-Menten equation can be used to derive a valueknown as the Michaelis-Menten constant (K_(M)) [See Biochemistry,Lehninger, 2nd Edition, pp. 189-192]. The equation expresses themathematical relationship between the initial rate of theenzyme-catalyzed reaction and the concentration of the substrate. Athigh substrate concentrations, the K_(M) of the glucose oxidase isexceeded and the reaction rate becomes nearly independently ofconcentration--this means that at such concentrations, it becomesdifficult to determine concentrations of glucose based on a ratereaction color change. In the glucose-glucose oxidase system, as thelevel of glucose present approaches 2 percent, the K_(M) of glucoseoxidase is exceeded, rendering it difficult to determine with accuracythe glucose level of the sample being tested.

Diabetic patients can have glucose levels ranging from 50 mg/dl (0.05%)to 10,000 mg/dl (10%). Because of this wide range, for detection andtreatment purposes, it is important to be able to quantitativelydetermine glucose levels in a range which encompasses about 1/2 up to 10percent. At present, dip-and-read reagent strips do not enabledetermination of glucose levels which exceed about 2 percent.

The present invention overcomes this limitation of dip-and-read reagentstrips and provides a method of measuring glucose levels of about 1/2 toabout 10 percent.

SUMMARY OF THE INVENTION

The present invention is directed to an enzymatic method for determiningthe glucose concentration in a test sample containing from about 1/2 to8 percent glucose. The method involves impregnating a carrier with anenzymatic testing composition which includes glucose oxidase, peroxidaseand a chromogen and drying the impregnated carrier. The carrier is thenimpregnated with from 0.5 to 1.5 percent by weight polystyrene. The testsample is then contacted with the test device, a detectable responseobserved and the glucose concentration is determined. The device of thepresent invention comprises a carrier matrix impregnated with anenzymatic testing composition containing glucose oxidase, peroxidase anda chromogen and subsequently impregnated with from 0.5 to 1.5 percent byweight polystyrene.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The carrier member used in the present invention can take on a multitudeof forms. It can be mono- or multi-phasic, comprising one or moreappropriate materials or mediums of similar or different absorptive orother physical characteristics. It can be hydrophobic or hydrophilic,bibulous or nonporous. It can take on many known forms such as thoseutilized for enzymatic reagent strips for solution analysis. Forexample, U.S. Pat. No. 3,846,247 teaches the use of felt, porous ceramicstrips, and woven or matted glass fibers. As substitutes for paper, U.S.Pat. No. 3,552,928 teaches the use of wood sticks, cloth, spongematerial, and argillaceous substances. The use of synthetic resinfleeces and glass fiber felts in place of paper is suggested in BritishPat. No. 1,369,139. Another British Pat. No. 1,349,623, suggests the useof a light-permeable meshwork of thin filaments as a cover for anunderlying paper carrier element. French Pat. No. 2,170,397 teaches theuse of carrier members having greater than 50 percent polyamide fiberstherein. Another approach to carrier members is disclosed in U.S. Pat.No. 4,046,513 wherein the concept of printing reagents onto a suitablecarrier is employed. U.S. Pat. No. 4,046,514 discloses the interweavingor knitting of filaments bearing reagents in a reactant system. All suchcarrier member concepts can be employed in the present invention, as canothers. Preferably the carrier member comprises a bibulous material,such as filter paper, whereby a solution or suspension of the glucoseoxidase is used to impregnate the carrier member.

It may be desirable to utilize a multi-step application of reagents. Insuch a case, two or more solutions or suspensions of reagents areprepared, the carrier member being dipped substantially into each withdrying steps between dippings. In such a case a porous material such aspaper might be most advantageous. Alternatively, it might be desirableto utilize a multiphasic carrier member, where two or more layers ofporous material are affixed one atop another. Still another approach tocarrier member incorporation is to sequentially coat a continuouspolymer with coatings containing different reagents of the immunoassaysystem. Filtering layers can be present in the carrier member topreclude potential interfering agents from reaching the assay system,while permitting access to any analyte present in the sample.

An indicated earlier, the carrier portion has incorporated thereinglucose oxidase, peroxidase and an indicator, e.g., a "benzidine-type"chromogen or substituted aniline chromogen or a combination thereof.Optionally, one or more water soluble polymers may be incorporated,e.g., polyvinyl pyrrolidone and a nonionic surfactant, e.g., apolyethoxylated fatty alcohol to provide more uniform color duringglucose testing. A suitable fatty alcohol sold under the tradedesignation Emulphor ON870, is available from GAF, New York, New York.

The carrier matrix can be impregnated with the enzymatic testingcomposition in several ways known to a person of reasonable skill in theart. One way is to pass a web of the carrier matrix material through animpregnating bath containing the testing composition ingredients so thatthe matrix becomes thoroughly saturated with impregnating solution. Thesaturated matrix is then dried, as in an air oven at 50° C., leaving thetest composition incorporated within the matrix.

Subsequent to impregnating the carrier matrix with the enzymatic testingcomposition, the matrix is impregnated with a solution of polystyreneand dried. The range of polystyrene which produces improveddetermination of glucose levels is from about 0.5 percent to 1.5 percent(weight/volume). A preferred amount of polystyrene is about 1 percent.

The following Examples illustrate the determination of glucoseconcentration according to the present invention.

EXAMPLE 1

Commercially available Eaton and Dikeman 204 filter paper was dippedinto an enzymatic testing solution having the following composition anddried for 15 minutes at 60° C.:

    ______________________________________                                        Sodium citrate buffer, 1.0M, pH 5.5                                                                    4.0    ml                                            Horseradish peroxidase, 3                                                                              4.0    ml                                            milligram/milliliter (mg/ml)                                                  Glucose oxidase, 5000 U/ml                                                                             0.06   ml                                            p-anisidine.HCl          320.0  mg                                            Distilled water          12.0                                                 ______________________________________                                    

The filter paper was impregnated with a 1.0 percent solution(weight/volume) of polystyrene (molecular weight ˜20,000) in toluene andagain dried. Test strips were prepared from the treated filter paper anddipped into urine which contained glucose concentrations ranging from0.0 to 5 percent. Control sample strips were prepared by impregnatinguntreated Eaton and Dikeman 204 filter paper with the above enzymatictesting composition, but without impregnating the paper withpolystyrene.

The performance of the test strips prepared as described above wasanalyzed instrumentally using a device known as the "Rapid Scanner".This device is a scanning reflectance spectrophotometer interfaced witha PDP-12 computer obtained from the Digital Equipment Corporation. Theinstrument is used for the rapid measurement of reflectance spectra inthe visual range. The computer allows for the storage of spectral dataand computations. Measurements of the performances of test strips in theRapid Scanner have the following advantages over visual observations.

1. The light source and conditions surrounding the sample remains fixed.In visual readings the light source can vary, not only in wavelengthcomponents, but also in relations to the locations of the strips beingobserved.

2. The detector characteristics remain fixed in the Rapid Scanner. Invisual observation, the detector (i.e. in the eyes of the observer)varies from person to person and, with the same person, from day to day.

3. The Rapid Scanner allows more precise quantitation of the data thandoes human observation, thereby permitting comparisons between theresults to be made in a more objective manner than with visualobservation.

The Rapid Scanner instrument was constructed by the Ames Division ofMiles Laboratories, Inc., Elkhart, Indiana, U.S.A., from whom completeinformation with respect to structural and performance characteristicsis obtainable.

Reflectance values obtained at 540 nanometers (nm) wavelength, after a60 second interval, are represented graphically in FIG. 1; where K/S isplotted against glucose concentration. K/S is defined as follows:##EQU1## in which K is a constant, S is the scattering coefficient ofthe particular reflecting medium, and R is the fraction of reflectancefrom the test strip. This relationship is a simplified form of thewell-known Kubelka-Munk equation [Gustav Kortum, "ReflectanceSpectroscopy", pp. 106-111, Springer-Verlaz, New York (1969)].

Slopes of segments of the K/S vs percent glucose carriers werecalculated, assuming linearity for the segments. The slopes obtained areshown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        K/S vs. Glucose Slope Values                                                    (%)Glucose                                                                           PolystyreneWith                                                                          PolystyreneWithout                                                                       ##STR1##                                       ______________________________________                                        1.0-2.0   0.41       0.27     65.8                                            2.0-5.0   0.28       0.08     28.8                                            ______________________________________                                    

As seen in FIG. 1 and as calculated in Table 1, at glucoseconcentrations ranging from about 1/2 to about 5 percent, thepolystyrene treated test strips have a greater slope than the untreatedtest strips. This greater slope indicates that in this glucoseconcentration range the reaction rate of the color change which occurson the urea-formaldehyde treated carrier is still dependent upon theglucose concentration.

The untreated test strips have a lesser slope, i.e. are becoming moreasymptotic at a faster rate in the range of about 1/2 to about 5 percentglucose concentration, indicating that the reaction rate of the colorchange which occurs on the untreated carrier is becoming moreindependent of glucose concentration, making it difficult to determineglucose concentration. The above example illustrates that the method ofthe present invention enables improved quantitation of the glucoseconcentration in a test sample within the range 1/2 to about 5 percentglucose.

As described below, another series of test strips was tested todetermine the upper range of glucose concentration which can be measuredaccording to the method of the present invention.

EXAMPLE 2

The commercially available filter paper described in Example 1 wasdipped into an enzymatic testing solution having the followingcomposition and dried for 15 minutes at 60° C.

    ______________________________________                                        Sodium citrate buffer, 1.0M, pH 5.5                                                                    2.0    ml                                            Horseradish peroxidase, 3 mg/ml                                                                        2.0    ml                                            Glucose oxidase, 5000 U/ml                                                                             0.01   ml                                            p-anisidine.HCl          400.0  mg                                            polyvinyl pyrrolidone (15%)                                                                            2.0    ml                                            Emulphor 0N870 (5%)      1.0    ml                                            Distilled water          3.0    ml                                            ______________________________________                                    

The filter paper was impregnated with a 1.0 percent solution(weight/volume) of polystyrene and the performance of the test stripsanalyzed as described in Example 1. Control sample strips were preparedas described in Example 1. K/S values were plotted against glucoseconcentration, and the slope of segments calculated, as summarized inTable 2 below.

                  TABLE 2                                                         ______________________________________                                        K/S vs. Glucose Slope Values                                                    (%)Glucose                                                                           PolystyreneWith                                                                          PolystyreneWithout                                                                       ##STR2##                                       ______________________________________                                        5.0-10.0  0.016      0.012    75.0                                            ______________________________________                                    

The results shown in Table 2 indicate that at glucose concentrationsranging from about 5 to 10 percent, the polystyrene treated test stripshave a greater slope than the untreated test strips. The above testresults, in conjunction with the test results shown in Example 1,indicate that the method of the present invention enables improvedquantitation of the glucose concentration within the range 1/2 to about10 percent glucose.

What is claimed is:
 1. In a method for determining the glucoseconcentration in a test sample containing from about 1/2 to 10 percentglucose which method includes the steps of; contacting said sample witha test device which includes a carrier matrix impregnated with anenzymatic testing composition including glucose oxidase, peroxidase, anda chromogen; and observing a detectable response produced by said testdevice; the improvement which comprises impregnating the carrier matrixwith a solution of from about 0.5 percent to 1.5 percent by weight ofpolystyrene subsequent to the impregnating of the carrier matrix withsaid testing composition.
 2. A method as claimed in claim 1 wherein thecarrier is paper.
 3. A method as claimed in claim 1 wherein polystyreneis present in an amount of about 1.0 percent by weight.
 4. A method asclaimed in claim 1 wherein the chromogen is a benzidine-type indicatoror a substituted aniline indicator.
 5. A method for determining theglucose concentration in a test sample containing from about 1/2 to 10percent glucose which comprises the steps of providing a test devicewhich includes a carrier having inpregnated therein an enzymatic testingcomposition comprising glucose oxidase, peroxidase, and a chromogen;subsequently impregnating the carrier with from 0.5 percent to 1.5percent by weight of a solution of polystyrene; contacting said testsample with the carrier of said test device; and observing a detectableresponse, whereby the glucose concentration is determined.
 6. A methodas claimed in claim 5 wherein the carrier is paper.
 7. A method asclaimed in claim 5 wherein the polystyrene is present in an amount ofabout 1.0 percent by weight.